Cystic Fibrosis (CF) is the most common inheritable lethal disease among Caucasians. There are approximately 25,000 CF patients in the U.S.A. The frequency of CF in several other countries (e.g., Canada, United Kingdom, Denmark) is high (ranging from 1 in 400 to 1 in 1,600 live births). There are numerous CF centers in the U.S.A. and Europexe2x80x94specialized clinical facilities for diagnosing and treating children and adolescents with CF.
Chronic respiratory infections caused by mucoid Pseudomonas aeruginosa are the leading cause of high morbidity and mortality in CF. The initially colonizing P. aeruginosa strains are nonmucoid but in the CF lung they inevitably convert into the mucoid form. The mucoid coating composed of the exopolysaccharide alginate leads to the inability of patients to clear the infection, even under aggressive antibiotic therapies. The emergence of the mucoid form of P. aeruginosa is associated with further disease deterioration and poor prognosis.
The microcolony mode of growth of P. aeruginosa, embedded in exopolysaccharide biofilms in the lungs of CF patients (Costerton et al., 1983), among other functions, plays a role in hindering effective opsonization and phagocytosis of P. aeruginosa cells (Pier et al., 1987; Pier 1992). Although CF patients can produce opsonic antibodies against P. aeruginosa antigens, in most cases phagocytic cells cannot effectively interact with such opsonins (Pressler et al., 1992; Pier et al., 1990; Pier 1992). Physical hindrance caused by the exopolysaccharide alginate and a functionally important receptor-opsonin mismatch caused by chronic inflammation and proteolysis are contributing factors to these processes (Pedersen et al., 1990; Tosi et al., 1990; Pier, 1992). Under such circumstances, the ability of P. aeruginosa to produce alginate becomes a critical persistence factor in CF; consequently, selection for alginate overproducing (mucoid) strains predominates in the CF lung.
Synthesis of alginate and its regulation has been the object of numerous studies (Govan, 1988; Ohman et al., 1990; Deretic et al., 1991; May et al., 1991). It has been shown that several alginate biosynthetic genes form a cluster at 34 min of the chromosome (Darzins et al., 1985), and that the algD gene, encoding GDP mannose dehydrogenase, undergoes strong transcriptional activation in mucoid cells (Deretic et al., 1987; 1991). GDP mannose dehydrogenase catalyzes double oxidation of GDP mannose into its uronic acid, a reaction that channels sugar intermediates into alginate production. The transcriptional activation of algD has become a benchmark for measuring molecular events controlling mucoidy (Deretic et al., 1991; Ohman et al., 1990; May et al., 1991). Studies of these processes have lead to the uncovering of several cis- and trans-acting elements controlling algD promoter activity: (i) The algD promoter has been shown to consist of sequences unusually far upstream of the mRNA start site (Mohr et al., 1990). These sequences (termed RB1 and RB2), as well as a sequence closer to the mRNA start site (RB3) are needed for the full activation of algD (Mohr et al., 1990; 1991; 1992). (ii) AlgR, a response regulator from the superfamily of bacterial signal transduction systems (Deretic et al., 1989), binds to RB1, RB2, and RB3, and is absolutely required for high levels of algD transcription (Mohr et al., 1990; 1991; 1992). (iii) Another signal transduction factor, AlgB, also contributes to the expression of genes required for alginate synthesis (Wozniak and Ohman, 1991). (iv) The peculiar spatial organization of AlgR binding sites imposes steric requirements for the activation process. The conformation of the algD promoter appears to be affected by histone like proteins [e.g. Alg (Hp1) (Deretic et al., 1992) and possibly IHF (Mohr and Deretic, 1992)], and perhaps by other elements controlling nucleoid structure and DNA topology. (v) The algD promoter does not have a typical xe2x88x9235/xe2x88x9210 canonical sequence (Deretic et al., 1989). It has been proposed that RpoN may be the sigma factor transcribing this promoter; however, several independent studies have clearly ruled out its direct involvement (Mohr et al., 1990; Totten et al., 1990). The present inventors have cloned and characterized a new gene; algU, which plays a critical role in algD expression (Martin et al., 1993). The algU gene encodes a polypeptide product that shows sequence and domainal similarities to the alternative sigma factor Spo0H from Bacillus spp. (Dubnau et al., 1988). Spo0H, although dispensable for vegetative growth, is responsible for the initial events in the triggering of the major developmental processes in Bacillus subtilis, viz. sporulation and competence (Dubnau et al., 1988; Dubnau, 1991). These findings suggest that activation of alginate synthesis may represent a cell differentiation process participating in interconversions between planktonic organisms and biofilm embedded forms in natural environments (Martin et al., 1993; Costerton et al., 1987).
Inactivation of algU abrogates algD transcription and renders cells nonmucoid, further strengthening the notion that algU plays an essential role in the initiation of mRNA synthesis at algD (Martin et al., 1993). algU maps in the close vicinity of muc markers that have been demonstrated in the classical genetic studies by Fyfe and Govan (1980) to cause the emergence of mucoid strains constitutively overproducing alginate. The mucoidy-causing property of muc mutations has been based on the ability of different muc alleles (e.g. muc-2, muc-22, and muc-25) to confer mucoidy in genetic crosses (Fyfe and Govan, 1980; 1983). The present application describes the presence of additional genes immediately downstream of algU, termed mucA and mucB, which also play a role in the regulation of mucoidy.
Detection of mucoid P. aeruginosa is a standard practice, however, due to the variability in expression of mucoidy on standard clinical media, more objective detection methods are needed. An early detection of conversion to mucoidy will be possible by using the present invention.
The present invention provides compositions and methods for the early detection and diagnosis of the conversion to mucoidy of Pseudomonas aeruginosa. The present invention also provides a molecular mechanism for the conversion from the nonmucoid to the mucoid state, including specific sequence alterations that occur in the mucA gene that cause the conversion and molecular probes for the early detection of this disease state.
xe2x80x9cRecombinant,xe2x80x9d as used herein, means that a protein is derived from recombinant (e.g., microbial) expression systems. xe2x80x9cMicrobialxe2x80x9d refers to recombinant proteins made in bacterial or fungal. (e.g., yeast) expression systems. As a product, xe2x80x9crecombinant microbialxe2x80x9d defines a protein produced in a microbial expression system which is essentially free of native endogenous substances. Protein expressed in most bacterial cultures, e.g., E. coli, will be free of glycan.
xe2x80x9cBiologically active,xe2x80x9d as used throughout the specification means that a particular molecule shares sufficient amino acid sequence similarity with the embodiments of the present invention disclosed herein to be capable of forming a algU-mucA-mucB complex, thereby repressing gene transcription from the algD promoter.
xe2x80x9cDNA sequencexe2x80x9d refers to a DNA polymer, in the form of a separate fragment or as a component of a larger DNA construct. Preferably, the DNA sequences are in a quantity or concentration enabling identification, manipulation, and recovery of the sequence and its component nucleotide sequences by standard biochemical methods, for example, using a cloning vector. Such sequences are preferably provided in the form of an open reading frame uninterrupted by internal nontranslated sequences. Genomic DNA containing the relevant sequences could also be used. Sequences of non-translated DNA may be present 5xe2x80x2 or 3xe2x80x2 from the open reading frame, where the same do not interfere with manipulation or expression of the coding regions.
xe2x80x9cNucleotide sequencexe2x80x9d refers to a heteropolymer of deoxyribonucleotides. DNA sequences encoding the proteins provided of this invention can be assembled from CDNA fragments and short oligonucleotide linkers, or from a series of oligonucleotides, to provide a synthetic gene which is capable of being expressed in a recombinant transciptional unit.
xe2x80x9cRecombinant expression vectorxe2x80x9d refers to a replicable DNA construct used either to amplify or to express DNA which encodes the fusion proteins of the present invention and which includes a transciptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a structure or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation initiation and termination sequences. Structural elements intended for use in yeast expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell. Alternatively, where recombinant protein is expressed without a leader or transport sequence, it may include an N-terminal methionine residue. This residue may optionally by subsequently cleaved from the expressed recombinant protein to provide a final product.
xe2x80x9cRecombinant microbial expression systemxe2x80x9d means a substantially homogeneous monoculture of suitable host microorganisms, for example, bacteria such as E. coli or yeast such as S. cerevisiae, which have stably integrated a recombinant transcriptional unit into chromosomal DNA or carry the recombinant transcriptional unit as a component of a resident plasmid. Generally, cells constituting the system are the progeny of a single ancestral transformant. Recombinant expression systems as defined herein will express heterologous protein upon induction of the regulatory elements linked to the DNA sequence or synthetic gene to be expressed.
The present invention provides a composition of matter comprising a first polynucleotide having the sequence of SEQ ID NO:10, a second polynucleotide complementary to the first polynucleotide or a polynucleotide differing from the first or second polynucleotide by codon degeneracy. Also claimed is a polynucleotide which hybridizes with the first or second polynucleotide, or an oligonucleotide probe for the first or second polynucleotide which hybridizes with said polynucleotide.
A further composition of matter of the present invention is a Pseudomonas aeruginosa mucA or mucB gene in substantially pure form. The mucA gene is defined as substantially comprising the sequence of FIG. 2 (SEQ ID NO:10) defined further as excluding the mucB coding region. Also claimed is a mucA gene defined further as differing from having an altered sequence. The alteration may be an insertion or deletion of at least one nucleotide, it may be a frameshift mutation, or a nonsense mutation. Alteration of the gene results in an inactive mucA gene product. In a preferred embodiment of the invention, the altered gene sequence has a deletion of nucleotide xe2x80x9cAxe2x80x9d from position 371 of the sequence of FIG. 2. In an alternatively preferred embodiment of the invention, the altered gene sequence has a deletion of nucleotide xe2x80x9cGxe2x80x9d from position 440 of the sequence of FIG. 2. In also a preferred embodiment of the invention, the altered gene sequence has an alteration of nucleotide xe2x80x9cCxe2x80x9d from position 362 of the sequence of FIG. 2.
The polynucleotide may be a polydeoxyribonucleotide or a polyribonucleotide. The oligonucleotide may be an oligodeoxyribonucleotide or an oligoribonucleotide. A further composition of matter is an oligonucleotide useful as a probe for and which hybridizes with the polynucleotide sequence of FIG. 2 or sequence alterations thereof. A preferred embodiment of the invention is an oligonucleotide comprising a sequence complementary to a region spanning the deletion at position 371. In particular, the oligonucleotide comprises the sequence 5xe2x80x2-GGGACCCCCCGCA-3xe2x80x2 (SEQ ID NO:2). The altered sequence of the mucA gene may have a deletion of the nucleotide xe2x80x9cGxe2x80x9d from position 439 or 440. One skilled in the-art would see that since there is a xe2x80x9cGxe2x80x9d in both positions 439 and 440 it is not possible to know which xe2x80x9cGxe2x80x9d is deleted in this altered sequence.
A further embodiment of the present invention is an oligonucleotide comprising a sequence complementary to a region spanning the deletion at position 439 or 440. In particular, this oligonucleotide comprises the sequence 5xe2x80x2-GAGCAGGGGCGCC-3xe2x80x2 (SEQ ID NO:3).
A further composition of matter of the present invention is a mucA gene having an altered sequence wherein the altered sequence is an insertion of nucleotides 5xe2x80x2-CAGGGGGC-3xe2x80x2 between positions 433 and 434. Also claimed is an oligonucleotide comprising a sequence complementary to a region spanning the insertion of the nucleotides 5xe2x80x2-CAGGGGGC-3xe2x80x2. A preferred embodiment is an oligonucleotide comprising 5xe2x80x2-CAGGGGGCCAGGGGGC-3xe2x80x2 (SEQ ID NO:4).
A further embodiment of the present invention is the use of these compositions of matter for a method of detecting conversion to mucoidy in Pseudomonas aeruginosa comprising detecting a loss of mucA or mucB function. In particular, a method of detecting conversion to mucoidy in Pseudomonas aeruginosa having an inactive mucA gene product comprising the detection of an altered sequence in the mucA gene is claimed. A preferred embodiment is a method of detecting conversion to mucoidy in Pseudomonas aeruginosa having an inactive mucA gene product comprising the detection of an altered sequence in the mucA gene. In this case, the altered sequence encodes an inactive product and the altered sequence is detected by hybridization with a complementary oligonucleotide. The complementary oligonucleotide may be 5xe2x80x2-GGGACCCCCCGCA-3xe2x80x2, 5xe2x80x2-GAGCAGGGGCGCC-3xe2x80x2, or 5xe2x80x2-CAGGGGGCCAGGGGGC-3xe2x80x2 (SEQ ID NOS:2-4). One skilled in the art could see that an altered sequence may comprise nucleotide changes, insertions, deletions or changes in the sequence leading to a nonsense or stop codon anywhere within this locus between about positions 300 and 500 of the sequence of FIG. 2.
A further embodiment of the present invention is a method of detecting conversion to mucoidy in Pseudomonas aeruginosa having an inactive mucA gene comprising the steps of: 1) obtaining Pseudomonas aeruginosa suspected of conversion to mucoidy to provide a test sample, and 2) hybridizing the test sample with an oligonucleotide 5xe2x80x2-GGGACCCCCCGCA-3xe2x80x2, 5xe2x80x2-GAGCAGGGGCGCC-3xe2x80x2, or 5xe2x80x2-CAGGGGGCCAGGGGGC-3xe2x80x2 (SEQ ID NOS:2-4). Positive hybridization indicates conversion to mucoidy in Pseudomonas aeruginosa. 
A further embodiment of the present invention is a Pseudomonas aeruginosa algU gene in substantially pure form. The algU gene comprises the sequence of FIG. 1.
The DNA sequences disclosed herein will also find utility as probes or primers in nucleic acid hybridization embodiments. As such, it is contemplated that oligonucleotide fragments corresponding to the sequence(s) of seq id no:1-14 and 20-23. For stretches of between about 10 nucleotides to about 20 or to about 30 nucleotides will find particular utility, with even longer sequences, e.g., 40, 50, 100, even up to full length, being more preferred for certain embodiments. The ability of such nucleic acid probes to specifically hybridize to algU, mucA and mucB-encoding sequences will enable them to be of use in a variety of embodiments. For example, the probes can be used in a variety of assays for detecting the presence of complementary sequences in a given sample. However, other uses are envisioned, including the use of the sequence information for the preparation of mutant species primers, or primers for use in preparing other genetic constructions.
Nucleic acid molecules having stretches of 10, 20, 30, 50, or even of 100 nucleotides or so, complementary to seq id no:1-14, 20-23, will have utility as hybridization probes. These probes will be useful in a variety of hybridization embodiments, such as Southern and Northern blotting in connection with analysing the complex interaction of structural and regulatory genes in diverse microorganisms and in clinical isoltaes from CF patients. The total size of fragment, as well as the size of the complementary stretch(es), will ultimately depend on the intended use or application of the particular nucleic acid segment. Smaller fragments will generally find use in hybridization embodiments, wherein the length of the complementary region may be varied, such as between about 10 and about 100 nucleotides, or even up to full length DNA insert of SEQ ID NOS:1-14, 20-23, according to the complementary sequences one wishes to detect.
The use of a hybridization probe of about 10 nucleotides in length allows the formation of a duplex molecule that is both stable and selective. Molecules having complementary sequences over stretches greater than 10 bases in length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained. One will generally prefer to design nucleic acid molecules having gene-complementary stretches of 15 to 20 nucleotides, or even longer where desired. Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as the PCR technology of U.S. Pat. No. 4,603,102 (herein incorporated by reference) or by introducing selected sequences into recombinant vectors for recombinant production.
Accordingly, the nucleotide sequences of the invention may be used for their ability to selectively form duplex molecules with complementary stretches of homologous, or heterblogous genes or cDNAs. Depending on the application envisioned, one will desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence. For applications requiring high selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids, e.g., one will select relatively low salt and or high temperatu target strand, and would be particularly suitable for isolating functionally related genes.
Of course, for some applications, for example, where one desires to prepare mutants employing a mutant primer strand hybridized to an underlying template or where one seeks to isolate specific mutant algU, mucA or mucB-encoding sequences from related species, functional equivalents, or the like, less stringent hybridization conditions will typically be needed in order to allow formation of the heteroduplex. In these circumstances, one may desire to employ conditions such as 0.15M-0.9M salt, at temperatures ranging from 20xc2x0 C. to 55xc2x0 C. Cross-hybridizing species can thereby be readily identified as positively hybridizing signals with respect to control hybridizations. In any case, it is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide, which serves to destabilize the hybrid duplex in the same manner as increased temperature. Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.
In certain embodiments, it will be advantageous to employ nucleic acid sequences of the present invention in combination with an appropriate means, such as a label, for determining hybridization. A wide variety of appropriate indicator means are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal. In preferred embodiments, one will likely desire to employ a fluorescent label or an enzyme tag, such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmental undesirable reagents. In the case of enzyme tags, calorimetric indicator substrates are known which can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with complementary nucleic acid-containing samples.
In general, it is envisioned that the hybridization probes described herein will be useful both as reagents in solution hybridization as well as in embodiments employing a solid phase. In embodiments involving a solid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to a selected matrix or surface. This fixed, single-stranded nucleic acid is then subjected to specific hybridization with selected probes under desired conditions. The selected conditions will depend on the particular circumstances based on the particular criteria required (depending, for example, on the G+C contents, type of target nucleic acid, source of nucleic acid, size of hybridization probe, etc.). Following washing of the hybridized surface so as to remove nonspecifically bound probe molecules, specific hybridization is detected, or even quantified, by means of the label.
Longer DNA segments will often find particular utility in the recombinant production of peptides or proteins. DNA segments which encode peptide antigens from about 15 to about 50 amino acids in length, or more preferably, from about 15 to about 30 amino acids in length are contemplated to be particularly useful, as are DNA segments encoding entire algU, mucA or mucB proteins. DNA segments encoding peptides will generally have a minimum coding length in the order of about 45 to about 150, or to about 90 nucleotides.
The nucleic acid segments of the present invention, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol. For example, nucleic acid fragments may be prepared in accordance with the present invention which are up to 10,000 base pairs in length, with segments of 5,000 or 3,000 being preferred and segments of about 1,000 base pairs in length being particularly preferred.
It will be understood that this invention is not limited to the particular nucleic acid and amino acid sequences of seq id nos:1-23. Therefore, DNA segments prepared in accordance with the present invention may also encode biologically functional equivalent proteins or peptides which have variant amino acids sequences. Such sequences may arise as a consequence of codon redundancy and functional equivalency which are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged.
DNA segments encoding a algU, mucA or mucB gene may be introduced into recombinant host cells and employed for expressing a algU, mucA or mucB structural or functionally related protein. Alternatively, through the application of genetic engineering techniques, subportions or derivatives of selected algU, mucA or mucB genes may be employed. Equally, through the application of site-directed mutagenesis techniques, one may re-engineer DNA segments of the present invention to alter the coding sequence, e.g., to introduce improvements to the antigenicity of the protein or to test algU, mucA or mucB mutants in order to examine transcription from the algD or related promoter activity at the molecular level. Where desired, one may also prepare fusion peptides, e.g., where the algU, mucA or mucB coding regions are aligned within the same expression unit with other proteins or peptides having desired functions, such as for immunodetection purposes (e.g., enzyme label coding regions).
As mentioned above, modification and changes may be made in the structure of algU, mucA or mucB coding. regions and still obtain a molecule having like or otherwise desirable characteristics. As used herein, the term xe2x80x9cbiological functional equivalentxe2x80x9d refers to such proteins. For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein""s biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence (or, of course, its underlying DNA coding sequence) and nevertheless obtain a protein with like or even counterveiling properties (e.g., antagonistic v. agonistic). It is thus contemplated by the inventors that various changes may be made in the sequence of algU, mucA or mucB proteins or peptides (or underlying DNA) without appreciable loss of their biological utility or activity.
In still further embodiments, the present invention concerns a method for identifying new compounds that inhibit transcription from the algD promoter, which may be termed as xe2x80x9ccandidate substances.xe2x80x9d Such compounds may include anti-sense oligonucleotides or molecule that encourage algU-mucA-mucB mediated repression of the algD promoter. The present invention provides for a method for screening a candidate substance for preventing P. aeruginosa conversion to mucoidy comprising: contacting the E. coli bacteria as described in Example 6 with an effective amount of a candidate substance; and assaying for reporter gene activity, wherein a decrease in the expression of the reporter gene indicates inhibition of algD promoter activity.